The GTP-dependent signal transduction systems have been found in many different cell types and enzyme systems. In vertebrate rod outer segments (ROS) illumination of rhodopsin triggers a cascade of reactions mediated by GTP-binding protein (transduction), which results in the hydrolysis of cGMP. In frog ROS, activation of phosphodiesterase (PDE) by light and GTP occurs as consequence of the release of an inhibitory moiety from the membrane bound PDE. Moreover, the hydrolysis of GTP is followed by the return of the GDP/G alpha (subunit of GTP- binding protein) complex to the membranes, as well as reassociation of the inhibitor with, and concomitant of PDE. However, these phenomena has not been detected in bovine ROS. Although hydrolysis of both cGMP and GTP has been studied intensively, the regulatory mechanisms of both cGMP synthesis and GTP formation remain unclear. Moreover, the linkage between PDE cascade and guanylate cyclase (cGMP synthesis) and/or guanine nucleotide metabolism, especially GTP formation, remains to be elucidated. Recent data suggest that cGMP synthesis and guanine nucleotide metabolism are closely synchronized with cGMP hydrolysis in ROS. The proposed experiments address purification and characterization of guanylate cyclase (synthesis of cGMP) and enzymes involved in guanine nucleotide metabolism (synthesis of GTP). Then, the proposal addresses to elucidate the regulatory mechanisms of these enzymes. Moreover, the mechanism for the linkage between these enzymes and the components of PDE cascade will be focused by using purified components of PDE cascade. The experiments have been designed biochemically to use the unique molecular properties of components in amphibian (frog and toad) rod outer segments. Some of these components have been purified and the presence of new regulators of both guanylate cyclase and enzymes for the synthesis of GTP has been detected in preliminary studies. The results of these experiments will reveal the new aspect of biological architecture which accomplishes the coordination of the many molecular species involved in the photoexcitation. These data will also provide the directions for the studies of GTP-dependent signal transduction, since the many common properties have been found in the different GTP-dependent signal transduction systems. Furthermore, this proposal, although basic studies, would be related to the pathological explanation of the visual abnormality.